University of Baghdad College of pharmacy

Aminopyrine and Antipyrine inhibitory studies on carbonic anhydrase

(CA), cholinesterases (ChEs) and monoamine oxidase (MAO)

A proposal submitted
In partial fulfillment of the requirements for the degree of Master of
pharmacy

Branch of pharmaceutical chemistry

:Prepared by
Shams Sabeeh Abdulraheem

2020
Introduction

Many researchers reported chemical and physical properties effects for various compounds for
human enzymes. Aminopyrine and antipyrine are (pyrazolone derivatives) are also known as
analgesic, anti-inflammatory, and antipyretic drugs [1]. Figure.1 shows the structures of
aminopyrine and antipyrine drugs [2].

Figure 1. Structures of Antipyrine(1) and Aminopyrine (2)

Heterocyclic compounds are important intermediate in medicinal and organic chemistry and that
makes them an important target for the treatment of a wide range of diseases and that enforce the
researchers for the design of new enzyme inhibitors [2].

Six enzymes present in this research study, two cholinesterases (ChEs); (acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE)), two Carbonic anhydrase isoforms (CA); (hCA I
and hCA II), and two monoamine oxidase enzymes; (MAO-A and MAO-B). The evaluation of
inhibitors will be done by depending on IC50 (it is the functional strength of enzyme inhibitor)
and Ki (it is the binding affinity of the enzyme inhibitor). Dixon plot will be used to estimate the
inhibition constant (Ki) of the inhibitors [3].

As well as, molecular docking calculations are present using the online docking server
SwissDock (EADockDSS, Swiss Institution of Bioinformatics, Switzerland, www.swissdock.ch)
this is a protein-ligand docking web service.

Literature review

Since the end of the 19th century, researchers interest in pyrazolone derivatives [4]. Compounds
possessing the pyrazolone derivatives have attracted much attention recently due to their
biological impact which includes, antibacterial, anti-inflammatory, hypoglycemic, antitumor, and
antitubercular [5],[6]. Many therapies have been achieved by inhibition of CA enzyme-like,
edema, Glaucoma, cancer, obesity, altitude sickness, neuropathic pain, and epileptic seizures.
Also, inhibition of AChE enzyme-like improves symptoms of Alzheimer's disease [7], as well as,
inhibition of MAO enzyme-like, they were used as antitumor agents with anti-estrogenic activity
[8-10].

Problem Statement

Develop enzyme inhibitors become essential and a big challenge recently. Evaluate the inhibitor
activity of many human enzymes using various compounds and their interaction is present in this
study. The biological impact of pyrazolone derivatives has attracted much attention lately. One
of the most important things is that little researches have been carried out on the CA, ChEs, and
MAO inhibition potential of pyrazolones derivatives; also the use of these inhibitors nowadays
became less popular due to the severity of their interactions with other drugs or certain foods.
Therefore, it is necessary to shed light on the design and improve pyrazolone compounds to treat
a wide range of disorders.

Hypothesis

The study investigates the ability of aminopyrine and antipyrine to inhibit CA, ChEs and MAO
to improve derivatives of a tertiary amino group depending on IC50 and Ki values and the
molecular docking for these drugs will be estimated through binding energy values (ΔG)
measured in (kcal/mol) for each ligand to each of the six protein models.

Research Objective

This research intends to investigate for: inhibitor activity of Aminopyrine and Antipyrine against
six enzymes, two cholinesterases (ChEs) include, acetylcholinesterase (AChE),
butyrylcholinesterase (BuChE), two Carbonic anhydrase isoforms(CA), (hCA I and hCA II) and
two monoamine oxidase enzymes; (MAO-A and MAO-B). Besides, molecular docking
investigate is present to study the possible interaction between protein and ligand during the
inhibition process.

Materials and Methods

The study will be organized according to the following steps:

Aminopyrine, Antipyrine, solvents, distilled water, AChE lyophilisate, BuChE lyophilisate,
Basford (1967) Reagents and all other compounds for inhibition assays can provide
commercially, two factors; nanomolar range will be depending on IC50 and Ki values and the
molecular docking for both drugs will be estimated through binding energy values (ΔG)
measured in (kcal/mol) for each ligand to each of the six protein models.

Laboratory equipment: glassware such as the beaker, reagent bottle, volumetric pipette,
volumetric flask, measuring cylinder, fume hoods, weighing scale, reagents, humidifier, stirrer
rod.

Apparatus used: Thermo Scientific Evolution 200 Series UV-VIS Spectrophotometer,

Gallenkamp apparatus (to measure the melting points), Shimadzu FT-IR 8101 PC infrared
spectrophotometer (to record IR spectra).

In all CA I, CA II, AChE, BuChE and MAO-A, MAO-B inhibition assay the test compound (1
mg) were dissolved in Dimethyl sulfoxide (1 ml) and diluted with distilled water then add
hydrazine hydrate (0.5mg) in the presence of catalytic amount of piperidine, the mixture refluxed
for 4 h then the solid product will be collected by filtration, dried and recrystallized from ethanol,
a control, and blank (no inhibitor, no enzyme respectively) measurement was recorded.
Molecular docking will be done using the online docking server SwissDock.

Biological and pharmacological evaluation: The study will be done on mitochondria, animals
(rats); all experiments will be performed in the morning according to the guidelines for the care
of laboratory animals, also fresh human blood will be received from a volunteer in
bioequivalence center of National center for drug control and research.

Elemental analysis will be made in the National center for drug control and research (NCDCR)
laboratories in Iraq-Baghdad, the study will begin in January and end on December/2021.

Statistical analysis

A significant difference among groups is assessed using two way ANOVA followed by post hoc
Tukey test to investigate whether there is a significant effect of the tertiary amino group in the
enhancing of the ability of inhibition of CA, ChEs and MAO inhibitors or not depending on
pyrazolone derivatives. Differences are considered significant at *p<0.01.
References

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